SYSTEMS AND METHODS FOR THE MONITORING OF PROTEIN COMPLEX FORMATION IN CELLS

摘要

Described herein is a bioluminescence resonance energy transfer (BRET) based system and method to monitor ternary complex formation in real-time in live cells with high sensitivity and accuracy. This system transfers energy simultaneously between a luciferase donor and intermediate and terminal acceptors, appropriately chosen to also enable transfer from the intermediate to terminal acceptor while minimizing contaminating signals. The system may also be adapted for quaternary complex detection by including a protein complementation assay (PCA) component. The system is broadly applicable to the detection of any protein ternary/quaternary complex such as those involving nuclear receptors, GPCRs, Receptor Tyrosine Kinase (RTKs), multimeric enzymes or structural proteins.

主权项

1. A biosensor for detecting a ternary protein complex comprising:
i. a first protein tagged with a Donor (D) protein, wherein D is a bioluminescent protein (Biol) having an emission spectrum (Biol-Em); ii. a second protein tagged with an Intermediate (I) protein, wherein I is a first Fluorescent protein (FP1) having an excitation spectrum (FP1-Ex) and emission spectrum (FP1-Em); and iii. a third protein tagged with an Acceptor (A) protein, wherein A is a second fluorescent protein (FP2) having an excitation spectrum (FP2-Ex) and an emission spectrum (FP2-Em);wherein
a) the FP1-Ex overlaps with the Biol-Em and overlaps minimally with the FP2-Ex; b) the FP1-Em overlaps with the FP2-Ex; c) the FP2-Ex overlaps with the Biol-Em; and d) the FP2-Em has a longer wavelength than the FP1-Em.